Directed Evolution of Enantiocomplementary Malonate Decarboxylases.

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Currently, the manufacture of pharmaceuticals, agrochemicals and other fine chemicals relies heavily on synthetic chemical methods, which use deleterious solvents, reagents and catalysts as well as non-renewable petrochemical precursors, which have serious detrimental environmental impact. Consequently, alternative biotechnology based processes are sought for the more economic and environmentally sustainable manufacture of those chemicals that are essential to maintain human health and quality of life. Central to the development of industrial biotechnologies is the availability of new enzymes, with tailored properties, that can be used to catalyse the transformation of renewable precursors into the required products under environmentally benign conditions. To date, industrial applications of enzymes have relied on a limited number of established enzymes, which catalyse a narrow range of transformations. However, recent genome sequencing has led to the discovery of a wider range of enzymes from natural sources. In addition new directed evolution technologies allow the properties of enzymes and even the reactions they catalyse to be altered and optimised for specific processes. Recently we solved the first structure and determined the detailed mechanism of a decarboxylase enzyme (AMDase) that catalyses the loss of carbon dioxide (decarboxylation) from malonic acid derivatives to generate chiral carboxylic acids. In this project, we aim to use these structural and mechanistic insights to develop more powerful decarboxylase enzymes that can provide access to a much wider range of structurally diverse carboxylic acids, which are particularly common intermediates in production of pharmaceuticals, agrochemicals and other products. The new decarboxylase enzymes are also attractive because the substrates can be generated from malonic acid, a natural precursor derived from renewable sources (fermentation). The availability of chiral carboxylic acids, which are single enantiomers (one of two possible stereoisomers that are non-superimposable mirror images) is of critical importance particularly for pharmaceutical production. Typically, enzymes only produce one of the two possible enantiomers, which is problematic if the opposite enantiomer is required. We will therefore use directed evolution technologies to develop enzymes that are enantiocomplementary. In this way, one enzyme can be used to produce one enantiomer (left-handed molecule), whilst another enzyme can produce the opposite enantiomer (right-handed molecule). Along with our industrial partners at BASF, who are the world's largest chemical manufacturers, we will tailor the new enantiocomplementary decarboxylases for production of key pharmaceutical intermediates. This includes chiral carboxylic acids used to manufacture non-steroidal anti-inflammatory drugs (from the ibuprofen family), the antiplatelet agent clopidogrel (the world's second-best selling drug) and captopril which is used to treat cardiac conditions. The family of enzymes to which the decarboxylases belong are known to be promiscuous, and can catalyse a wider range of reactions than decarboxylations, including racemisations and isomerisations. We aim to further explore the promiscuity of this enzyme family, with a view to developing alternative reactions that would also be of industrial importance.